Abscisic acid is a naturally occurring plant hormone which acts primarily to inhibit growth of plants, maintain dormancy of buds, inhibit fruit-ripening, activate the pathogen resistance response defense, induce senescence in already-damaged cells and their proximate neighbors, and help the plant tolerate stressful conditions. See Arteca, R. (1996), Plant Growth Substances: Principles and Applications. New York: Chapman & Hall; Mauseth, J. D. (1991), Botany. An Introduction to Plant Biology. Philadelphia: Saunders. pp. 348-415; Raven, P. H., Evert, R. F., and Eichhorn, S. E. (1992), Biology of Plants. New York: Worth. pp. 545-572.
Abscisic acid owes its name to the belief that this plant growth regulator causes the abscission of leaves from deciduous trees in the fall. Absicin II and dormin are names previously used for this plant hormone. The chemistry and physiology of abscisic acid and its analogs is described by Milborrow, Ann. Rev. Plant Physiol. 1974, 25, 259-307.
Abscisic acid analogs are structural derivatives of 2-cis-, 4-trans-(S)-(+)-abscisic acid. An extensive series of analogs of abscisic acid has been prepared by researchers at the Plant Biotechnology Institute of the National Research Council of Canada, Saskatoon, Saskatchewan. Some of these abscisic acid analogs are disclosed in U.S. Pat. Nos. 5,201,931, 5,518,995 and 6,004,905, which are incorporated herein by reference. Presently preferred abscisic acid analogs include PBI-376, PBI-524, PBI-697 and PBI-410.
Prior art (U.K. Pat. No. 1251867 and Railton and Wareing, Planta 112, 65-69, 1973) taught, inter alia, preparation of amine salts of racemic abscisic acid. A salt of racemic (R,S)-(±)-2-trans-,4-trans-abscisic acid with the chiral alkaloid brucine was prepared as a means of resolving a small quantity of the racemate in order to study the physical properties of its enantiomers (J. C. Bonnafous, et al., Tetrahedron Letters, 1119-1122, 1973). Salts of (S)-(+)-abscisic acid are disclosed in co-pending U.S. patent applications Ser. No. 12/011,846 filed Jan. 30, 2008 entitled SALTS, AQUEOUS LIQUID COMPOSITIONS CONTAINING SALTS OF S-(+)-ABSCISIC ACID and Ser. No. 12/011,845 filed Jan. 30, 2008 entitled SALTS, AQUEOUS LIQUID COMPOSITIONS CONTAINING SALTS OF ABSCISIC ACID ANALOGS AND METHODS OF THEIR PREPARATION, and in a co-pending patent application No. 61/083,202 filed on the same day as this application. Contents of these patent applications are herein incorporated by reference. However, these patent applications do not disclose salts of abscisic acid analogs having substantially enhanced biological activity relative to 2-cis-,4-trans-(S)-(+)-abscisic acid itself, nor do they disclose salts of abscisic acid analogs having substantially enhanced biological activity relative to the abscisic acid analogs themselves.
As noted above, abscisic acid analogs are carboxylic acids, and thus in a medium having an acidic pH, they are protonated and in their neutral undissociated form. This uncharged, undissociated form is more lipophilic than a salt of an abscisic acid analog, and penetration of the uncharged acid form into the plant cuticle would be favored relative to the charged, dissociated form of the abscisic acid analog present at higher pH (Blumenfeld and Bukovac 1972, Planta 107: 261-268). The uncharged, undissociated form of the abscisic acid analog would be expected to cross cell membranes from the apoplast into the cytosol more easily than a salt form would. In spite of this, we have surprisingly found that treatments comprising certain salts of abscisic acid analogs of this invention have substantially better biological activity when compared with similar treatments comprising the acid form of the same abscisic acid analog at the same concentration. We have also surprisingly found that certain iodide salts, when applied to plants in combination with the same abscisic acid analog, produce substantially enhanced biological activity.
Abscisic acid was first defined in the early 1960s as a growth inhibitor accumulating in abscising cotton fruit and in leaves of sycamore trees photoperiodically induced to become dormant. See, Finkelstein R R, Rock C D (2002), Abscisic Acid Biosynthesis and Response, The Arabidopsis Book: Vol. 45, No. 1 pp. 1-48. Since then, abscisic acid has been shown to regulate many aspects of plant growth and development, including embryo maturation, seed dormancy, germination, cell division and elongation, etc. Although abscisic acid has historically been thought of as a growth inhibitor, young tissues have high abscisic acid levels, and abscisic acid-deficient mutant plants are severely stunted because their ability to reduce transpiration and establish turgor is impaired. Exogenous abscisic acid treatment of mutants restores normal cell expansion and growth.
Abscisic acid is thought to initiate its effects on cells through binding to receptor proteins, although their identities and locations are still largely unknown. Activation of the putative receptor(s) causes a chain of events that results in rapid changes in ion channels and slower changes in the pattern of gene transcription. While many individual components of this chain of events have been identified, a complete picture has not yet been obtained.
Commercial formulations comprising abscisic acid are used in agriculture for various purposes, such as improving stress tolerance, slowing growth rate, adjusting flowering phase, and other purposes. Abscisic acid has also been reported to possess insect inhibition qualities. See U.S. Pat. Nos. 4,434,180 and 4,209,530 to Visscher. Abscisic acid in a powdered form is currently commercially available from Lomon Biotechnology Company, Ltd., a Chinese company, which markets it as a substance that, among other uses, improves the yield and quality of crops.
However, one of the problems associated with preparation of formulations of abscisic acid analogs is their relatively poor solubility in water: The solubilities of these compounds in water are even lower than that of (S)-(+)-abscisic acid itself, and for (S)-(+)-abscisic acid not more than about 3 grams per liter or alternatively, less than 0.3% by weight will dissolve at ordinary temperatures. Stated differently, a concentration of about 3000 parts per million (ppm) is the highest concentration of (S)-(+)-abscisic acid that can be achieved in pure water at room temperature, and the maximum for abscisic acid analogs is even lower. While abscisic acid analogs have better solubility in some organic solvents, liquid formulations of abscisic acid analogs in organic solvents are unacceptable in some contexts because of flammability, toxicity, or pollution considerations. For example, the Environmental Protection Agency of the U.S. state of California is currently requiring that liquid formulations of agricultural products contain no volatile organic solvent, and several other U.S. states are considering similar regulations. Nonvolatile organic solvents have the detriment that, since they do not evaporate, they remain in the agricultural product as it impinges upon and is absorbed into the target plant, with a probability of causing phytotoxicity and contaminating food products, since the amount of the solvent greatly exceeds the amount of active ingredient applied.
A further problem observed with concentrated solutions of abscisic acid analogs in organic solvents is that it is difficult to prepare more dilute solutions by dilution into water without having a portion of the abscisic acid analog precipitate out in a gummy form that redissolves only very slowly and with great difficulty. This is of practical importance because a major use of abscisic acid analogs in agriculture or horticulture is for the reduction of transpiration in nursery plants being prepared for transplantation or for sale to consumers, for which purpose the abscisic acid analog may be applied by means of an injection system and automatic or hand applicators. The solution for use in such an applicator must be a concentrate between about 50 and 100 times more concentrated than the dose rate that is actually reaching the plants when they are treated by foliar spray or drench. Thus for a typical application to the nursery plants of 60 to 600 ppm, the concentrate must contain between 3000 and 60,000 ppm of (S)-(+)-abscisic acid in a solution that will mix instantly and completely with the water flowing through the hose, in such a way that there is no possibility of formation of a precipitate that would clog the nozzle through which the water containing active ingredient is applied to the plants or the growing media of the plants. As explained above, the solubility of abscisic acid analogs in water is less than 3000 ppm at ordinary ambient temperature, so such an intermediate solution cannot practically be prepared in water. A solution of an abscisic acid analog in an organic solvent cannot be used in such an injection applicator, because precipitation of the active ingredient will occur during the mixing into the water flowing in the system, and the spray nozzle will be clogged. Because of the solubility limitation, it is also not possible to provide a liquid formulation of the abscisic acid analog in organic solvent at a higher concentration (e.g. 10%) and then at the time of application to prepare an intermediate dilution in water to achieve the desired concentration of 3,000 to 60,000 ppm in the reservoir of the injection applicator.
An identical problem arises in the case of application of an abscisic acid analog to a vineyard, orchard or agricultural field through an irrigation system, a practice commonly known as chemigation. Again, such a system requires a concentrated solution of the active ingredient in a liquid solvent in such a form such that that solution is instantly and completely miscible with a stream of water flowing through the irrigation system. If any precipitation were to occur, it would block the nozzles (known as emitters) through which the water and dissolved active ingredient reach the target plants. Again in this situation a formulation consisting of an organic solution of the abscisic acid analog would not be acceptable because of the problem of the low water solubility.
Abscisic acid analogs are very expensive. They can be manufactured only by multi-step chemical synthesis, involving costly reagents and several laborious purifications. When these abscisic acid analogs are applied to plants, uptake is poor, so a large excess must be employed. It is possible to improve uptake of the abscisic acid analogs by combining them with various surfactants; however, it is well known that the use of surfactants can damage the foliage, flowers and fruits of sensitive plants, producing phytotoxicity and reducing the value or destroying the crop.
While powdered formulations of abscisic acid analogs could be prepared, it is often more convenient to use concentrated liquid solutions instead of powders. Therefore, there is an unmet need in the art for highly efficacious abscisic acid analog formulations comprising salts of abscisic acid analogs, which are much more soluble in water than the acids themselves are and do not require that surfactants be employed to promote their uptake by plants.